Vaccine and serum for endotoxin associated disease immunization and treatment, detoxified endotoxin, and bacterial mutant

ABSTRACT

A combination vaccine, with methods of preparation and treatment, for protection against Gram negative bacterial diseases. The vaccine includes a killed suspension of a bacterial mutant from the taxonomic family Enterobacteriaceae, a B-lymphocyte proliferating immune modulator, and a protein and lipid binding adjuvant. A hyperimmune serum for treating diseased animals is prepared by vaccinating a donor animal with the combination vaccine and then preparing clot serum containing antigen specific antibodies. The mutant is ATCC No. 53000 and is produced by ionizing radiation of Salmonella enteritidis. The immune modulator is a detoxified extract of lipopolysaccharide and is useful with many antigens to enhance primary immune response.

REFERENCE TO THE PRIOR APPLICATION

This application is a continuation of Ser. No. 07/279,338 filed Dec. 2,1988, now abandoned, which is a continuation of Ser. No. 06/697,008filed Jan. 31, 1985, now abandoned, which and is a continuation-in-partof Ser. No. 06/597,115 filed Apr. 5, 1984 now abandoned entitled VACCINEAND SERUM FOR ENDOTOXIN ASSOCIATED DISEASE IMMUNIZATION AND TREATMENT.

BACKGROUND OF THE INVENTION

This invention relates to a vaccine and serum for immunization against,and treatment for, gram negative bacteria diseases. More specifically,this invention relates to a bacterial mutant of Salmonella enteritidisand its use in a combination vaccine to immunize mammals and birdsagainst diseases caused by endotoxin producing gram negative bacteria inthe taxonomic family Enterobacteriaceae. This invention also relates toa detoxified endotoxin immune modulator useful in the treatment ofanimals and men in combination with other antigens.

In the field of animal husbandry, endotoxin associated diseases poseserious animal health problems and consequently, represent an economicinfluence of significant proportion.

In horses, endotoxin-associated diseases include founder (i.e.,laminitis), colic (i.e., abdominal crisis associated with dietaryengorgement and other stressful phenomena such as abdominal obstruction,intestinal ischemia, Gram negative bacterial enteritis/diarrhea,intestinal malabsorption, transport stress, parturition, etc.) septicarthritis, and Gram negative intrautrine infections.Endotoxin-associated diseases in cattle include laminitis in both dairyand feedlot cattle, sudden death syndrome in feedlot cattle, mastitis indairy cattle, and dysentery, white scours or colibacillosis, andSalmonella diarrhea in baby calves. Endotoxin-associated disease inswine include parturition dysagalactia (i.e., mammary gland failurerelated to Gram negative endometritis), intestinal edema disease, andbaby pig Salmonella diarrhea. Salmonella diarrhea, hemorrhagicsepticemia, infection of the air sacs and sinuses; and fowl cholera andother Pasteurelloses are examples of endotoxin-associated diseases ofbirds.

Previous treatment for endotoxin mediated and/or associated diseases hasbeen retrospective (i.e., after development of clinical illness) and hasbeen limited to cbemotherapeutic intervention. Prevention measures werenot achieved with such treatment. Prior limited, definitive, vaccinalprotection from Gram negative septicemia and/or endotoxemia has beenaccomplished only via (a) individualized vaccines comprised ofautogenous bacterial isolates expressing various antigenic epitopes(K-antigens or O-carbohydrate side chains) or (b) live vaccinescomprised of attenuated or deletion-modified, live bacterial isolates.

The major disadvantage of the current methodologies for treatingendotoxin mediated and/or associated diseases is that such treatmentsare initiated only after clinical illness has developed, whichfrequently is after the disease has attained an irreversible state. Theprior vaccinal protection for Gram negative septicemia and/orendotoxemia that has been reported for individualized vaccines comprisedof autogenous bacterial isolates is not time, cost or productionefficient because such vaccines are produced retrospectively, afterdisease has developed.

The primary disadvantages of the polyvalent vaccines comprised ofmultiple bacterial isolates expressing various antigenic epitopes(K-antigens or O-carbohydrate side chains) are that the bacterialisolates causing disease at any given time are subject toepidemiological shifts and/or drifts in antigenic epitopes causing achange in antigenic specificity and thus loss of protective efficacy.The K-antigens or O-carbohydrate side chains also are potent stimulatorsof immunoglobulin IgE which is responsible for undesirable anaphylactoidreactions in many animal species, especially the horse.

The primary disadvantages of live vaccines comprised of attenuated ordeletion-modified bacterial isolates is that they have the potential forreversion to the wild-type parential strains and thus resumption ofpathogenicity for vaccinated animals.

Accordingly, a long felt need exists for a vaccine and serum to immunizeand treat against diseases caused by Gram negative bacteria and toovercome the deficiencies found in the prior art. One principal objectof this invention is to meet this need.

SUMMARY OF THE INVENTION

An immune modulator which is non-toxic to mammals, including man, hasbeen developed which, when used with another antigen to immunize suchmammals, enhances rapid and elevated antibody responses.

A combination vaccine and byperimmune serum was developed for protectionof horses and cattle against endotoxin-associated diseases. The vaccinealso has potential for protecting other animals such as swine and sheep,and birds against similar bacterial and/or endotoxin-associateddiseases.

The vaccine comprises a killed suspension of a non-O-carbohydrate sidechain bacterial mutant, an immune modulator with propensity forB-lymphocyte proliferation and a carrier having high lipophilic andproteinophilic affinity. For immunization, the vaccine is administeredintramuscularly or subcutaneously at concentrations having at least1×10⁷ bacteria and 100 micrograms immune modulator. For treatment of ananimal having a Gram negative bacterial caused disease, serum isprepared from a vaccinated donor and administered to provide aprotective level of antibodies.

A mutant of a Salmonella organism is especially effective as thebacterial mutant and a method of making such mutant is disclosed.

A principal object of the invention is to provide a detoxified endotoxinimmune modulator useful in enhancing antibody responses when inoculatedinto a mammalian host and a method of making and using such immunemodulator.

Still a further object of the invention is to provide a bacterial mutantof a Salmonella organism useful alone and in combination with saidimmune modulator in the vaccination of animals against Gram negativebacterial diseases.

Another object of the invention, accordingly, is to provide a safe,effective and economical vaccine for protection against Gram negativebacterial diseases.

Still another object of the invention is to provide a method forpreparing a safe, effective and economical vaccine for protectionagainst Gram negative bacterial diseases.

Yet another object of the invention is to provide a method forimmunization against Gram negative bacterial diseases.

An additional object of the invention is to provide a method fortreating an animal infected with a Gram negative bacterial disease.

A further object of the invention is to provide a serum for the safe,effective and economical treatment of an animal infected with Gramnegative bacteria.

Other and further objects of the invention together with the features ofnovelty appurtenant thereto, will appear in the course of the followingdescription.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves several different concepts, namely, (1) abacterial mutant, specifically a non-O-carbohydrate side chain bacterialmutant from the family Enterobacteriaceae; (2) a vaccine featuringbacterial mutant and a protein and lipid binding carrier and preferablyincluding an immune modulator which is a detoxified endotoxin; (3) theaforesaid immune modulator and the method of preparing it, the immunemodulator being useful in many applications, including man, and incombination with different active ingredients to increase the speed andamount of antibody response in the person or animal being treated; (4) ahyperimmune serum for treating Gram negative bacterial diseases and amethod of using said serum.

The concept of broad spectrum protection via a combination vaccine perse, and/or combination vaccine-elicited hyperimmune serum againstbacteremias and/or endotoxemias mediated and/or associated with a widevariety of Gram negative bacteria is an economical breakthrough for theanimal industry using new molecular concepts in applied immunology.Diseases for which protection is developed by the combination vaccineincludes those associated with endotoxin disseminated intravascularcoagulation and, more particularly, the Gram negative bacteriaSalmonella enteritidis, Salmonella typhimurium, Salmonella typhosa,Salmonella minnesota, Salmonella abortus-equi, and Escherichia coli.

The development of protection accrues from a combination vaccine of (a)a bacterial mutant with broad protective potential; (b) an immunemodulator with specific propensity for B-lymphocytes and, consequently,the earlier occurrence of higher levels of neutralizing and opsonizingantibodies of high antigenic affinity and avidity and; (c) a carrier ofhigh lipophilic and proteinophilic affinity, thus insuring uniformcomponent suspension and prolonged antigenic release. The hyperimmuneserum, derived from the combination vaccine, offers a new treatmentmode, heretofore unavailable for clinical use.

Other aspects of our invention include the combination of the mutant anda carrier alone which is effective, but not on the whole as effective asthe three component vaccine which includes the immune modulator.

The immune modulator itself is prepared by detoxifying extractedlipopolysaccharide. It is useful in the vaccine of this invention andalso in admixture with other active ingredients, particularly antigens,to enhance the speed and amount of antibody responses and potentiatesproduction of wide ranges of immunologic specificities. The immunemodulator is non-toxic to man and other mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting immune response over time of the bacterincompared to bacterin+endotoxoid preparation.

FIG. 2 is a graph comparing pre-immune and hyperimmune globulin of horse#23 using a passive immunization mouse model.

FIG. 3 is graph comparing pre-immune and hyperimmune globulin of horse#24 using a passive immunization mouse model.

MUTANT

The mutant is deposited in the American Type Culture Collection as ATCCNo. 53000. The date of deposit is Jan. 16, 1985, 12301 Parklawn DriveRockville Md., 20852.

The parent isolate used to prepare the genetically modified MutantStrain R-17 was isolated from an active diarrheal infection of a horseat the University of Missouri College of Veterinary Medicine. Theoriginal clinical isolate was isolated on MacConkeys Agar, exhibiting alactose negative, smooth, mucoid glistening colony of 3.5-4 mm diameterat 24 hours incubation at 37 C. Biochemical analysis using an API System(API Laboratory Products, 200 Express St., Plainview, N.Y. 11803) inconjunction with the API Profile Recognition System and characterizationon routine laboratory media identified the original clinical isolate asSalmonella enteritidis (Serotype B-typhimurium). This organism isdescribed by Ewing and Martin. (Ewing, W. H. and W. J. Martin:Enterobacteriaceae. In Manual of Clinical Microbiology, 2nd ed.Washington, American Society for Microbiology, 1974).

A specific embodiment of the organism of this invention relates to adeletion mutant strain of the parent isolate of Salmonella enteritidis(Serotype B-typhimurium) effected by ionizing radiation. Ionizingradiation, by virtue of high energy penetrance, induces free radicalformation which labilizes cytoplasmic molecules causing single-strandedbreaks in the deoxyribonucleic acid molecules, thus resulting in a highfrequency of deletion mutations. Surviving mutants frequentlyphenotypically express various degrees of inability to synthesize intactlipopolysaccharide. Such mutants are easily recognized, since theyexhibit smaller diameter, flat, rough (R) colonies, in contrast tolarge, punctate or convex, smooth (S) colonies produced by the parentbacterium.

X-ray mutagensis was accomplished on standard pour plates seeded withviable parent bacteria. Plates were irradiated in 5 second increments toa maximum of 35 seconds using a Machelett OEG 60 X-ray tube withberyllium window, operated at 50 kV peak and 25 mA, to give a dose rateof 250 rad/sec. Irradiated plates were held at 4 C for 2-4 hours andthen incubated in the dark at 37 C to preclude Photoreactivation. At theend of 24 hours incubation plates were examined for a change in colonialmorphology. Colonies of equal or less than 2 mm diameter exhibitingrough (R) morphology were selected, passaged at least 10 times on solidplate media, and passaged at least 3 times by intraperitonealinoculation of laboratory mice to insure stable rough (R) phenotypicexpression. Mutant strain R-17 was assayed for avirulence, in comparisonwith the parent isolate by a standard mouse potency assay viaintragastric inoculation. Purified lipopolysaccharide from the mutantstrain R-17 and the parent isolate were analyzed chemically byelectrophoresis in 2% sodium dodecyl sulfate--10% polyacrylamide gels(Palva, E. T & P. Helena Makela, 1980, Lipopolysaccharide Heterogeneityin Salmonella typhimurium, Analyzed by Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis, European Journal of Biochemistry107:137-143) and biologically by the chromatogenic limulus lysate assay(Webster, C. J. 1980, Principles of a Quantitative Assay for BacterialEndotoxins in Blood That Uses Limulus Lysate and a ChromogenicSubstrate, Journal of Clinical Microbiology 12(5):644-650), andseroagglutination (Lindberg, A. A. & L. Le Minor. 1984, Serology ofSalmonella. Vol. 15, pp. 1-141; In Methods in Microbiology, T. Bergan,Editor, Academic Press, New York, 1984) and no O-carbohydrate antigencould be detected. Therefore, the mutant strain R-17 was presumed to bea Chemotype I or II, naked-core mutant and a novel embodiment of thisinvention.

Trichloroacetic Acid Extraction (Boivin Method) of Lipopolysaccharide

Lipopolysaccharide (LPS) is extracted from either acetone dried bacteriaor wet bacteria suspended in 5 volumes of distilled H₂ O with 0.25Naqueous Trichloroacetic Acid. The solubilized LPS (supernatant) isseparated from residual bacteria (pellet) by centrifugation (5000×g, 30min, 40° C.). The pH of the supernatant is adjusted to pH 6.8 with 1ONNaOH and LPS then precipitated from the supernatant by the addition of 2volumes of cold absolute ethyl alcohol. The precipitated LPS iscollected and washed (3×) with cold absolute ethanol by centrifugation(10,000×g, 1 hr, 4° C.), lyophilized, and stored at 4° C. until use.

Preparation of the Immune Modulator

Trichloroacetic acid extracted lipopolysaccharide (LPS) is dissolved in100 volumes of freshly prepared pyridine, 90% formic acid (2:1 v/v) byslowly increasing the temperature to the boiling point and holding forapproximately 15 minutes or until apparent clearing. Detoxification thenis accomplished by the addition of an equal volume of distilled H₂ O tothe LPS-pyridine-formic acid solution and refluxing for 60 minutes. Thedetoxified LPS is precipitated overnight (4° C.) by the addition of 4volumes of cold absolute ethanol and centrifugation (10,000×g, 1 hr, 4°C.), washed (3×) with cold absolute ethanol and then lyophilizeddetoxified LPS immune modulator was stored at 4° C. or reconstituted in0.1% aqueous triethylamine for immediate use in effecting immunepotentiation.

Potentiation of Immune Response & Hybridoma Fusion with Immune Modulator

Purified lipopolysaccharide is known to effect lymphocyte blastogensisin vitro, i.e., cultured tissue cells. In man and other mammalslymphocytes produce the interleukins (IL-1, IL-2) which mediate theimmune response and thereby the production of antibodies, via theecosatetraenoic acid metabolites (i.e. prostanoids or prostaglandins).It is also known that purified lipopolysaccharide potentiates IL-1 andprostacylin synthesis in vivo. However, purified lipopolysaccharide orin situ (associated with Gram negative bacteria per se)lipopolysaccharide possess intact O-carbohydrate side chain antigenswhich are toxic when introduced in vivo (i.e. into mammals), causinguntoward febrile responses, coagulopathies, and sometimes fataldisseminated intravascular coagulation via anaphylactoid reactions inthe sensitized host. Purified lipopolysaccharide also is cidal tomammalian tissue or cells grown in vitro culture, at picagram and lownanogram concentrations.

Embodiments of this invention include: 1) a novel method for preparationof an immune modulator which is non-toxic to mammals and tissue cellcultures, 2) a novel method employing the immune modulator forimmunizing mammals to either particulate or soluble antigens whichenhances more rapid and greatly elevated antibody responses and alsopotentiates recognition of broader spectra of antigenic determinants(epitopes) and consequent production of wider ranges of immunologicspecificities, and 3) a novel method for enhancing the frequency ofhybridization between antibody synthesizing-plasmacytoma cells andB-lymphocytes in cell culture from 15-30% to greater than 85% by primaryimmunization of donor mammals with particulate or soluble antigens inthe presence of immune modulator.

Immune modulator, when given simultaneously with antigen, enhances theprimary immune response of C57BL/6J mice. Enhancement occurs both withparticulate antigens such as Pseudomonas aeruginosa and with solubleantigens such as keyhold limpet hemocyanin. Animals injected withantigen and immune modulator simultaneously demonstrated higher antibodytiters at 7, 14, and 35 days after injection than did animals receivingantigen alone. Immune modulator not only enhances antibody titers earlyin the immune response, but more importantly, appears to prolong highserum antibody levels. Enhancement of the specific antibody response byimmune modulator is not significantly affected by route of injection,since enhancement is observed when antigen and immune modulator areinjected intravenously, intraperitoneally, or subcutaneouly inincomplete Freund's Adjuvant.

These results are summarized in Table A. In these experiments 5 femaleanimals were in each group. Experimental animals received antigen andimmune modulator (75 μg/dose) and controls received an equal amount ofantigen and sterile saline. Serum antibody titers were measured by anindirect ELISA assay.

Immune modulator demonstrates no toxicity as assayed by its effect oncell culture growth. When SP2/0 mouse myeloma cells were cultured withimmune modulator at concentrations of 100, 10, 1, 0.1 ng per ml ofculture fluid, cultures with immune modulator reached cell densitiesequivalent to or slightly greater than the corresponding controlcultures. Myeloma cells were cultured in RPMI 1640 media supplementedwith 10% fetal bovine serum 2%,L-glutamine, 1% sodium pyruvate, andantibiotics. Immune modulator was added at appropriate concentrations tothe media as a sterile, aqueous solution. Cell densities were determinedat 24, 48, 72, and 96 hours after the addition of immune modulator or anequivalent volume of sterile distilled water.

                                      TABLE A                                     __________________________________________________________________________    Immunization of C57BL/6J Mice with Particulate or Soluble Antigens with       and without Immune Modulator by Various Routes of Inoculation.                                              Antibody Titers                                                       Route of                                                                              After Immunization                              Animal Group                                                                            Antigen     Inoculation                                                                           7 days                                                                             14 days                                                                            35 days                               __________________________________________________________________________    Immune Modulator                                                                        P. aeruginosa                                                                             Intravenous                                                                           *1:32,000                                                                          1:128,000                                                                          1:32,000                              Control   P. aeruginosa                                                                             Intravenous                                                                            1:8,000                                                                           1:16,000                                                                           1:4000                                Immune Modulator                                                                        P. aeruginosa                                                                             Subcutaneous                                                                           1:4,000                                                                           1:16,000                                                                           1:16,000                                        in incomplete Freund's                                              Control   P. aeruginosa                                                                             Subcutaneous                                                                           1:2,000                                                                           1:4,000                                                                            1:2,000                                         in incomplete Freund's                                              Immune Modulator                                                                        hemocyanin  Intraperitoneal                                                                        1:16,000                                                                          1:256,000                                                                          1:64,000                              Control   hemocyanin  Intraperitoneal                                                                        1:4,000                                                                           1:64,000                                                                           1:16,000                              __________________________________________________________________________     *Dilution of Sera                                                        

The Vaccine

The vaccine comprises a bacterial mutant (bacterin), an immune modulator(endotoxoid) and a protein and lipid binding carrier (adjuvant). Thevaccine is administered intramuscularly or subcutaneously atconcentrations equal to or greater than 1×10⁷ bacteria (preferably,1×10¹⁰ bacteria), 100 or greater micrograms (preferably 100 to 4000micrograms) detoxified endotoxin, in a lipophilic-proteinophilicabsorbent carrier.

The bacterin preferably consists of a killed suspension of anon-O-carbohydrate-side chain mutant of Salmonella enteritidis. Thebacteria may be prepared by inoculation of sterile, enriched broth witha subculture of the Salmonella enteritidis mutant and aerobic incubationat 37° C. to obtain maximal bacterial mass. The bacteria are killed byaddition of a bactericidal agent such as Merthiolate. The bacteria arechecked for non-viability, then washed (4×) with sterile, non-pyrogenicphystologic saline, and reconstituted to the desired stock concentrationfor admixing with the other components of the vaccine.

The detoxified endotoxin is prepared by admixing Gram negative bacterialendotoxin to pyridine-formic acid (2:1) is solution. Theendotoxin-pyridine-formic acid mixture is thoroughly mixed in a sterilereflux condenser apparatus, the temperature increased to the boilingpoint and refluxed to obtain optimal methylation of endotoxin. Themethylated, detoxified endotoxin then is precipitated from the aqueousreflux mixture by the addition of alcohol, collected by centrifugation,washed by resuspension in alcohol and recentriguation, and, finally,dissolved in nonpyrogenic distilled water to the desired stockconcentration for admixing with the other components of the vaccine.

The adjuvant consists of high affinity lipophilic and proteinophiliccarrier sufficient to absorb the protein-moiety of the bacterin andlipid-moiety of the detoxified endotoxin. Preferably the adjuvant is afatty acid based adjuvant, oil based adjuvant, or alum based adjuvant(such as dialuminum trioxide). The carrier properties of dialuminumtrioxide function by maintaining uniform suspension and allow prolongedrelease of the bacterin and detoxified endotoxin, thus insuring maximalantibody production. When tit aluminum trixoide is utilized as the highaffinity proteinophilic and lipophilic adjuvant, approximately 1.5% byvolume is optimal.

Normal horses when immunized with the combination vaccine developedantibodies in their blood stream sufficient for protection whenchallenged in the laboratory by overfeeding with carbohydrate (i.e.dietary engorgement mimicking what occurs in nature) or intravenousinjection of bacterial endotoxins (i.e., artificially induced diseasemimicking what occurs in nature). Normal cattle when immunized with thevaccine developed antibodies; and exhibited no rise in body temperature,or abnormal increase in numbers or types of white cells. The advantagesof the combination vaccine include (a) the mutant bacterium which isdevoid of components normally present in its cell wall which causeundesirable anaphylactoid reactions; (b) an adjuvant or carrier whichinsures prolonged release of bacterin and/or detoxified endotoxin andconsequently maximal production of neutralizing antibodies, and (c) thedetoxified endotoxin which itself enhances the production of an earlierand higher level of the desirable neutralizing antibodies against intactendotoxin and bacteria. The combination vaccine, also has the quality ofproviding broad spectrum protection against many Gram negative bacterialdiseases, since the basic structure of the antigen is common to mostGram negative bacteria; yet is devoid of those components present inexisting vaccines which cause undesirable anaphylaxis.

Laboratory observations indicate that carbohydrate overload causesincreased concentrations of acid in the gut (i.e., large bowel) , whichin turn damages the normally impermeable bowel lining and simultaneouslydecreases the number of Gram negative bacterial normally present in thecontents and lining of the bowel by enhancement of migration into theblood stream (septicemia) and acid killing in the gut per se. Thekilling of these bacteria, results in the release of endotoxin fromtheir cell walls, which in turn, also crosses the acid-damaged gutlining into the blood stream. The endotoxin in the blood stream thencauses undesirable blood clots in the small blood vessels (intravascularcoagulation). In the case of horses and other hoofed animals, theseundesirable blood clots form in the small blood vessels, ultimatelycausing death of the hoof tissue, permanent crippling and/or death ofthe animal.

The specific advantages of the combination vaccine in horses is toprevent the crippling and killing effects of founder or colic, byneutralizing endotoxin and/or Gram negative bacteria that gets into theblood stream of horses experiencing accidental overfeeding, grassfounder, or stress. The specific advantage of the combination vaccine incattle also is to prevent or reduce the endotoxin associated diseases byneutralizing any endotoxin and/or Gram negative bacteria that gainentrance to the blood stream or other tissues. Sudden death syndrome infeedlot cattle, mediated by endotoxin from Gram negative bacteria of gutorigin, is a classic example of such a disease with current, immenseeconomic implications in the cattle industry.

Potency and Safety of Vaccine Components

Extensive testing has been conducted to establish the potency and safetyfor the components of the vaccine.

It was discovered that incorporation of the immunoregulator endotoxoidwith bacterin would elicit an earlier, and enhanced immuneresponsiveness in vaccinated animals. Groups of healthy adult horses andponies were vaccinated intramuscularly with bacterin+endotoxoid or thebacterin alone. Blood samples had been drawn on the animals a weekbefore the start of the experiment and immune profiles indicated allwere normal (i.e., no history of chronic laminitis). Serum samples werecollected at 24 hours intervals after immunization, and antibody titersascertained using the antigen-specific, solid-phase radioimmunoassay.The data presented in FIG. 1 indicates that animals vaccinated with thebacterin+endotoxoid developed detectable antibody titers as soon as 3days after vaccination compared to 7 days for those animals receivingthe bacterin alone. Examination of the immune response curves in FIG. 1between days 3 and 14 indicated a steeper slope for thebacterin+endotoxoid groups compared to the bacterin-only groups. Thisindicated an enhanced rate of antibody production for the former.Consequently, it was postulated that the bacterin+endotoxoid groupsdeveloped earlier protection. Additionally, an overall greater degree ofprotection resulted due to higher concentrations of neutralizing and/oropsonizing antibodies in their circulation, compared to those animalsreceiving the bacteria alone.

Endotoxoid (i.e., detoxified endotoxin) was administered to mice, horsesand ponies, and cattle to ascertain the maximal amount of endotoxoidthat could safely be incorporated with the bacterin, as animmunoregulator in the vaccine. An LD₅₀ for CF-1 mice usually isattained within 72-96 hours by intravenous injection of 0.3-0.6 mg ofnative endotoxin.

Groups of male, CF-1 mice (15-20 grams) were inoculated in the marginaltail vein with 0.1 milliliter physiologic saline, 300 μg of endotoxin in0.1 of physiologic saline, and respective concentrations of 600 μg, 6000μg, and 2,000 μg of endotoxoid in 0.1 milliliter of phystologic saline.The mice were observed at 24 hour intervals for adverse effects andmortality. Deaths were observed in the endotoxin (positive control)group by 48 hours with maximal mortality (56%) recorded at 72 hours(Table 1). In comparison, no mortality was observed for the groupreceiving twice (600 ug) that amount of endotoxoid; only 17% mortalityoccurred in the group receiving the 20× (6000 μg) endotoxoid; while 40×(12,000 μg) of endotoxoid resulted in an LD₅₃. It was concluded that theendotoxoid was at least forty times less toxic than its nativeendotoxin.

                  TABLE 1                                                         ______________________________________                                        Comparison of Endotoxoid and Endotoxin in CF-1 Mice                           Groups of CF-1                                                                              Concentrations of                                               Mice receiving:                                                                             μg in 0.1 ml, IV                                                                         Survival                                          ______________________________________                                        Endotoxin     300           44                                                Placebo       Physiologic Saline                                                                          100                                               Endotoxoid                                                                    Conc. 1       600           100                                               Conc. 2       6,000         83                                                Conc. 3       12,000        47                                                ______________________________________                                    

Adult horses and ponies were inoculated intramuscularly with up to 5 mgof endotoxoid. The animals were observed twice daily for 4 days forpyrogenicity, loss of peripheral perfusion, elevated heart rate, bloodpressure, lethargy, and diarrhea. None of the animals receiving the 2.5mg dose exhibited any adverse symptomology. A few of the animalsinoculated with the 5 mg dose experienced transient rise in temperatureto 103°-105° F., slight elevation in heart rate and mild loss ofperipheral perfusion. These symptoms subsided within 8-12 hours. None ofthe animals developed diarrhea, blood dyscrasias or other irreversibleeffects. It was concluded that up to 2.5 mg (2500 μg) , or 25 times the100 μg of endotoxoid proposed for incorporation as an immunoregulator inthe vaccine, could be safely used in horses and ponies.

Cattle were inoculated intramuscularly with up to 1000 μg of endotoxoidand observed at 2 day intervals for 16 days for pyrogenicity, leukopeniaand/or leukocytosis, mononuclear cell abnormalicies (differentialcounts), erythrocyte abnormalicies, lethargy, and diarrhea. N untowardeffects were observed in the cattle. It was concluded that up to 1000 μgof endotoxoid could be safely incorporated as an immunoregulator in thevaccine for cattle.

Safety of the Vaccine

The safety of using the vaccine was evaluated relative to the followingcriteria: 1) Large (up to 5×) doses in laboratory mice, horses andcattle relative to recommended dosage for routine vaccination regimens;2) Inoculation of horses and cattle with multiple doses within a shorttime span; 3) The route of inoculation, (i.e., intramuscuiar,subcutaneous or intraperitoneal); 4) In laboratory mice as a means offuture quality control; 5) In laboratory horses and ponies, wheremultiple criteria could be evaluated; and 6) In field studies, wherehorses and ponies and cattle in large numbers and of ubiquitous genepool could be evaluated for a limited number of parameters.

Groups of adult, mixed sex mice (20-25 gm) were inoculated with 1 mlaliquots of the vaccine or components of the vaccine. The mice wereobserved for mortality, hair coat texture, spinal arching and clusteringindicative of peripheral vascular hypothermia, dehydration, lethargy,diarrhea, and abscessation for up to 96 hours.

                  TABLE 2                                                         ______________________________________                                        Inoculation of Mice with Vaccine                                              Group            96 Hour Survival (%)                                         ______________________________________                                        vaccine/subcutaneous                                                                           100                                                          vaccine/intramuscular                                                                          *100                                                         vaccine/intraperitoneal                                                                        **70                                                         bacterin/subcutaneous                                                                          100                                                          endotoxoid/subcutaneous                                                                        100                                                          carrier/subcutaneous                                                                           100                                                          carrier/intraperitoneal                                                                        ***50                                                        physiological saline                                                                           100                                                          ______________________________________                                         *Mice were inoculated intramuscularly with 100.1 ml aliquots due to small     size.                                                                         **Mice died of severe dehydration due to adsorption of serum proteins by      the highly lipoproteinophilic carrier.                                        ***Subsequent intraperitoneal inoculation of 1 ml aliquots, (equivalent t     quantity of carrier in 2 ml of vaccine).                                 

The data in Table 2 Indicated that 1 ml doses of the vaccineadministered either subcutaneously or intramuscularly offered no risk tolaboratory mice. However, the peritoneal route of inoculation resultedin 30% mortality due to the high ratio of lipo-proteinophilic carrier tobody mass (1:20) and consequent protein dehydration. It was concludedthat the adverse effect observed for intraperitoneal Inoculation oflaboratory mice would be Irrelevant since the target species for thevaccine would Involve phenomenally greater body weight to carrierratios, and the recommended route of inoculation would be intramuscularor subcutaneous rather than intraperitoneal.

Healthy, adult horses were Inoculated intramuscularly with 2 consecutive5× (5×10¹⁰ bacteria, 500 μg endotoxoid) doses of the vaccine, six daysapart. The animals were observed daily for 20 days for anorexia,lethargy, diarrhea, dehydration, and tenderness, swelling and/orabscessation at the injection site. The only adverse effect wastransient tenderness and swelling at the injection site which subsidedwithin 48-72 hours.

Cattle weighing 500-650 lbs. were vaccinated intramuscularly with 2consecutive 4.5× (equivalent to 4.5×10¹⁰ bacteria and 437 μg endotoxoid)doses of vaccine 18 days apart. One cow was vaccinated with one 11.25×(equivalent to 1.125×10¹⁰ bacteria and 1 mg endotoxoid) dose of thevaccine. All animals were examined twice daily for anorexia, lethargy,diarrhea, dehydration, and tenderness, swelling and/or abscessation atthe injection site. No adverse reactions were observed In any of thecattle.

The above data indicated that the dose range and regiment recommendedfor use in horses and cattle offered no untoward risk to the animals.

Healthy, adult horses were inoculated intramuscularly with 2.5×(equivalent to 2.5×10¹⁰ bacteria and 200 μg endotoxoid) doses of thevaccine at 3 day intervals for 9 days, allowed to rest for 7 days andthen the 3 dose-3 day interval inoculation regimen was repeated for 5additional times. The horses received a total of 18 inoculations over aperiod of 64 days. Consecutive inoculations were alternated from theneck to buttock. The animals were observed daily for anorexia, lethargy,diarrhea, dehydration; and tenderness, swelling and/or abscessation atinjection sites. The only adverse reaction was localized tenderness,circumscribed swelling and abscess formation at two injection sites inone animal after ten inoculations. The abscesses, upon drainage, rapidlyresolved and the inoculation regimen was continued. The animalsexperienced no other adverse reactions.

Calves weighing between 190-650 lbs. were inoculated subcutaneously with4× (equivalent to 4×10¹⁰ bacteria and 400 μg endotoxoid) doses ofvaccine. Seventeen days later a portion of the calves were re-inoculatedintramuscularly with a 1× dose of the vaccine. All animals were observeddaily for 120 days for anorexia, lethargy, diarrhea and dehydration. Allanimals were examined at 3 day intervals for 15 days, followed by 30 dayintervals for 120 days thereafter, for weight loss, body temperature,and blood dyscrasias. Five of the calves had firm, circumscribed nodulesin the subcutaneous tissue which resorbed by 8-12 days after the primary(4×) subcutaneous inoculation. Similar nodules were inapparent afterre-inoculation of the same calves with 1× doses by the intramuscularroute.

A herd of horses and ponies were inoculated intramuscularly with 2 dosesof vaccine (equivalent to 1×10¹⁰ bacteria and 100 ug endotoxoid per kgbody weight) 14 days apart. Animals were observed daily for 20 days foranorexia, lethargy, diarrhea, dehydration and tenderness, swellingand/or abscessation at injection site. No untoward effects, other than amild degree of transient tenderness at the injection site in a fewanimals, were observed.

A herd of feeder cattle weighing between 550-650 lbs. was inoculatedwith 1 dose of vaccine (equivalent to 1×10¹⁰ bacteria and 100 μgendotoxoid per kg body weight). A portion of the cattle were Inoculatedintramuscularly and the balance were inoculated subcutaneously. Thecattle were observed daily for 50 days for anorexia, lethargy, diarrhea,add dehydration, tenderness, swelling and/or abscessation at injectionsites. No adverse reactions were observed related to the injectionsites. No apparent systemic abnormalicies were observed related to thevaccination. No subcutaneous nodulation was apparent in any of thesubcutaneously inoculated animals.

In summary, it is apparent that intramuscular or subcutaneousinoculation of cattle, and horses or ponies with the vaccine atreasonable and recommended dose-regimens offers little or no risk toanimals.

Efficacy of Vaccine

Healthy, adult horses and ponies were immunized by intramuscularinoculation of two 1× (equivalent to 1×10¹⁰ bacteria and 100 μgendotoxoid) doses of vaccine approximately two weeks apart. The animalswere bled at approximately weekly intervals and antibody titersascertained by radioimmunoassay. Immune response curves usually reacheda maximum approximately 20-30 days after the primary immunization or10-20 days after the secondary or anamnestic immunization. Protectiveefficacy was determined by either carbohydrate engorgement (Per Os) oradministration of sublethal doses of endotoxin (intravenously) tovaccinated animals at various times after the secondary immunization.Seventy to eighty percent of non-vaccinated horses or ponies developedObel grade 3-4, acute laminitis by forty to fifty hours aftercarbohydrate engorgement with a cornstarch-wood flour gruel administeredvia stomach tube at the dosage of 17.6 gram gruel per kilogram bodyweight. One hundred percent (100%) of non-vaccinated horses or poniesdeveloped tachypnea, dyspnea and ataxia within 2-3 minutes and passedfluid, non-formed stools by 45 minutes after intravenous administrationof endotoxin at dosage of 10 ug per kilogram body weight.

                  TABLE 3                                                         ______________________________________                                        Challenge of Vaccinated Horses by Carbohydrate                                Overload and IV-Endotoxin                                                                     Combination                                                   Challenge       Vaccine    Placebo                                            ______________________________________                                        CHO - (Per Os)  *2/19 (10.5%)                                                                            75-85%                                             Endotoxin (IV)  *3/8 (37.5%)                                                                             100%                                               Total           *5/27 (18.5%)                                                 ______________________________________                                         *Number of animals that developed Obel grade (3-4) laminitis or               endotoxinmediated symptomology after challenge.                          

Table 3 indicates that approximately 90% of the vaccinated animals,compared with 15 to 25% of the non-vaccinated-control pool (consistingof 100 animals over 12-14 years) failed to develop Obel grade 3-4laminitis after challenge by carbohydrate overload, suggesting at leasta 65-75% protective efficacy. Similar comparison of the vaccinated andnon-vaccinated horses challenged with sublethal endotoxin, indicates agreater than 60% protective efficacy. Consequently, it was concludedthat vaccination of normal, adult horses or ponies with two 1× doses ofvaccine resulted in protection of up to 90% of the animals fromcarbohydrate-induced laminitis (i.e., founder) and greater than 60% fromendotoxin-induced endotoxemia.

A group of cattle of mixed age and sex were vaccinated subcutaneouslywith one or two doses of vaccine. Animals were bled and sera obtained at3 day intervals for 15 days, and then at 30 day intervals for up to 120days. Antibody titers were determined by radioimmunoassay. All animalshad developed 2 to 4 fold increases in antibody titer by 20 days aftervaccination. Detectable titers were present in approximately 70% of theanimals at 120 days after vaccination. A portion of the group was placedon a high carbohydrate ration thirty days after vaccination and after 17weeks had not developed any signs of anorexia, diarrhea, lameness orsudden death syndrome.

A herd of feeder cattle weighing between 550-680 lbs. were vaccinatedwith 1-(1×) dose of vaccine and monitored daily for diarrheal disease,lameness and sudden death. No diarrheal disease, lameness or mortalityoccurred during 11 weeks of observation.

The Serum

The development and therapeutic use of hyperimmune serum was based onthe rationale that non-vaccinated animals with clinically apparentendotoxin-associated diseases are not afforded the time necessary fortheir own immune systems to build protective levels of antibodies, aftervaccination with the combination vaccine. Thus, passive immunizationwith pre-existing, stored antibodies developed by another animal(hyperimmune serum) provided a means of short-term protection that couldaid in amelioration of the endotoxin-associated disease until theanimal's own immune system was sufficiently protective. An acutelaminitic episode in the non-vaccinated horse that accidentally getsinto the grain bin and subsequently founders, is a classic example withcurrent implication in the horse industry.

The hyperimmune serum is comprised of clot serum or plasma, or partsthereof (gamma globulin, immunoglobulin, or immunoglobulin IgGT) whichcontain antibody(s) specific for the core component(2-Keto-3-deoxyoctonic Acid-Lipid A) in endotoxin of bacteria in thetaxonomic family Enterobacteriaceae which are elicited byhyperimmunization of animals with the combination vaccine.

Hyperimmune sera is prepared by intramuscular injection of healthy adulthorses with 6 consecutive 2.5 ml doses of the combination vaccine at 3day intervals followed by 2 consecutive 2.5 ml doses at 7 day intervals.Serum samples are taken from the horses prior to vaccination and at 3day intervals thereafter for serologic analyses. Concentrations ofantigen-specific immunoglobulins (IgG, GT, A and M) are determined byradio-immunoassay using ¹²⁵ I-Protein A. When each animal's immuneresponse has reached a high-titer plateau, 12 liter quantities of wholeblood are collected via vena puncture. The hyperimmune sera is obtainedby centrifugation after coagulation (approximately 24 hr.), and thenheat inactivated (56° C., 30 min.) add stored at 4° C. until use orsubsequent purification of gamma globulin or immunoglobulin.

Gamma globulin is prepared by precipitation from aliquots of hyperimmunesera with 50% saturated ammonium sulfate (SAS). The precipitate is thenresuspended in 0.01M phosphate buffer (PB, NaH₂ PO₄ --Na₂ HPO₄, pH 8)and exhaustively dialyzed against the same buffer to remove the SAS.

The gamma globulin obtained from 50 ml aliquots of hyperimmune sera isabsorbed onto a column (5×50 cm) of diethylaminoethyl (DEAE) celluloseequilibrated with PB, pH 8. The column is developed initially withequilibrating buffer (PB, pH 8) to elute IgG, followed by the additionof a NaCl gradient (00.3M) to the PB to disassociate IgG(T). The eluateis collected in 5 ml aliquots using a refrigerated fraction collectorand elution peaks monitored continuously using a Beckman DB-GTSpectrophotometer and dual wavelength of 360 and 380 nm. Proteinconcentration is determined using the Warberg-Christian constant andconfirmed by the Lowry method. The IgG(T) aliquots are pooled,lyophilized and stored at -40° C. for subsequent use in passiveimmunization. Horses, experimentally foundered by overfeeding withcarbohydrate or intravenous injection of bacterial endotoxins showedrapid improvement upon administration of hyperimmune serum, obtainedfrom other horses vaccinated with the combination vaccine. Similarly,death from intravenous injection of bacterial endotoxins is precluded inlaboratory mice by immunization with hyperimmune serum obtained fromhorses vaccinated with the combination vaccine.

Safety, Potency and Efficacy of Hyperimmune Serum

Groups of healthy, male adult (20 gm), (CF-1) mice were inoculated,intraperitoneally (IP) with 1 ml aliquots of 100%, 10% or 0.1% of whole,equine hyperimmune serum on four (4) consecutive days. The animals wereobserved for four days at 12 hour intervals for dyspnea, rigor, nose andtail perfusion, swollen eyes, coat texture and death. No untowardeffects were observed from the IP serum injections.

A healthy adult pony was inoculated with 700 ml of hyperimmune serumadmixed with 1000 ml of Lactated Ringers Solution, by intravenous (IV)drip over 90 minutes. The animal was .monitored for 61/2 hours (at 10-15minute intervals) for change in body temperature, heart rate, peripheralperfusion; and discomfort and/or distress. The pony experienced no signsdiscomfort or stress. The body temperature and heart rate exhibitedslight (statistically insignificant) increases (100.4°→101.3° F.;40→beats/min.) approximately 60-90 minutes after initiation of theIV-drip.

In a clinic trial, a 900 lb. horse with complications associated fromseptic shock was Inoculated Intravenously with 1200 ml hyperimmune serumcontinuously over a period of 10 hours in Lactated Ringers solution. Theanimal exhibited no untoward signs of toxicity and indeed showed markedimprovement.

To evaluate the safety of intramuscular inoculation, healthy, adultponies were inoculated intramuscularly (IM) with 0, 10, 20, and 40 ml ofhyperimmune serum. The animals were monitored at 30 min., 1, 2, 4, 8,16, and 24 hours after inoculation for (1) urination; (2) diarrhea; (3)rigors; (4) peripheral perfusion; (5) temperature; (6) respiration; (7)heart rate; (8) leukocytosis (or -penia); and (9) erythrocytosis (or-penia). No untoward effects were observed. A diffuse nodule wasapparent in the neck of one pony administered the 40 ml doseintramuscularly which had receded by 4 hours.

Gamma globulin, containing the protective antibody, was extracted fromthe hyperimmune sera in order to evaluate protective efficacy on amilligrams-protein basis. Pre-immune and hyperimmune sera fromindividual horses hyperimmunized with vaccine were compared byinoculating subsets of CF-1 mice with various concentrations of gammaglobulin in divided doses on two consecutive days before intravenouschallenge with endotoxin. The data in FIGS. 2 and 3 compare pre-immuneand hyperimmune globulin from two separate horses (#23 and #24) usingthe passive immunization mouse model. Hyperimmune globulin was preparedfrom horse #23 at two subsequent times after hyperimmunization (#23A,#23B). Comparison of the percentages of mice surviving 96 hours afterendotoxin challenge that were passively Immunized with 50 ug or more of423 pre-immune or hyperimmune (#23A and #23B) globulin indicates atleast a 20 (for #23B) to 50 (for #23A) percent increase in survival forthose subsets receiving hyperimmune globulin (FIG. 2). Comparison of #24pre-immune with #24 hyperimmune also Indicates similar protectiveefficacy but to a lesser degree (FIG. 3). It was concluded that thehyperimmune serum contains antibodies which can passively protect micefrom lethal endotoxin challenge.

The efficacy of the hyperimmune gamma globulin was ascertained bychallenge (endotoxin or carbohydrate engorgement) of subsets of horsesand ponies after intravenous inoculation with 5, 15, or 20 mg antibodyprotein/kg body weight. All animals received a mixture of #23A and #23Bhyperimmune gamma globulin. Combination of the two preparations wasnecessary in order to insure adequate quantities of the known antibodyprotein to complete the studies. Protection was defined as the markeddelay and/or amelioration of the immediate vital sign changes, anddevelopment of equal to or less than Obel Grade 2 disease, in sublethalendotoxin and/or carbohydrate challenged animals.

                  TABLE 4                                                         ______________________________________                                        Passive Immunization of Horses with Pre-Immune                                and Hyperimmune Serum                                                                 Passive Immunization with                                             Challenge Hyperimmune globulin                                                                          Pre-Immune globulin                                 ______________________________________                                        CHO - (Per Os)                                                                          40%             100%                                                Endotoxin (IV)                                                                           0%              50%                                                ______________________________________                                         Percentage figures represent developing Obel grade (3-4) laminitis or         endotoxinmediated symptomology after challenge.                          

Conclusion

The advantage of the combination vaccine is that it is prophylactic innature, in opposition to current treatment modes which are initiatedretrospectively or only after development of disease. An added advantageof the combination vaccine is that immunized animals develop an earlierand higher degree of protection from many of the endotoxin associateddiseases without the risks inherent in existing vaccines such as (a)provoking potentially fatal anaphylaxis; (b) developing potentiallyfatal infections by reversion of live, non-pathogenic bacterins topathogenic forms; or (c) losing ability to elicit protection because ofrelative changes in strains of bacteria causing disease. The detoxifiedendotoxin component of the combination vaccine, as a potent immunemodulator with propensity for B-lymphocytes causes not only more rapidproliferation of these antibody progenitor cells, but also their earlieroccurrence in the activated functional state, thus resulting inproduction of protective levels of antibody in the host's circulationmuch sooner after vaccination than observed for conventional bacterinvaccines. The bacterin component of the combination vaccine, comprisedof a mutant exhibiting a naked core antigen (2-Keto-3-deoxyoctonicAcid-Lipid A), devoid of any of the O-carbohydrate side chains (KAntigens) present in conventional bacterin vaccines, precludes thedevelopment of O-carbohydrate specific Immunoglobulin E (IgE, Reagin)and thus elicitation of IgE-mediated anaphylaxis after vaccination.Since the naked core antigen, in contrast to O-carbohydrate side chains(K-antigens serotypes), is common to many Gram negative bacteria, itelicits antibodies of broad cross-protection, while also precluding theloss of protective efficacy because of epidemiological shifts and driftsin O-carbohydrate side chains (K-antigens, serotypes) relative to time.

Prior to the development of the combination vaccine and hyperimmuneserum elicited by the combination vaccine, medical management involvedprimarily chemotherapy only after onset of the Endotoxin-associateddiseases. The advantage of hyperimmune serum, is that in thenon-vaccinated animal with clinically apparent disease, it mayameliorate the disease process, thus precluding crippling and/or deathin the horse or other species.

From the foregoing, it will be seen that this invention is one welladapted to attain all the ends and objects hereinabove set forthtogether with other advantages which are obvious and which are inherentto the structure.

It will be understood that certain features and subcombinations are ofutility and may be employed without reference to other features andsubcombinations. This is contemplated by and is within the scope of theclaims.

Since many possible embodiments may be made of the invention withoutdeparting from the scope thereof, it is to be understood that all matterherein set forth or shown in the accompanying drawings is to beinterpreted as illustrative and not in a limiting sense.

Having thus described the invention, we claim:
 1. A broad spectrumvaccine for immunization of animals against Gram negative bacterialdisease comprising:a) a killed suspension of non-O-carbohydrate sidechain bacterial mutant ATCC 53000 as a bacterin, b) a detoxifiedlipopolysaccharide immune modulator having specific propensity forB-lymphocytes to cause rapid proliferation of antibody progenitor cellsand their earlier occurrence, thus resulting in enhanced production ofprotective and neutralizing antibodies in a host's circulation againstintact endotoxin and bacteria, and c) a protein and lipid bindingcarrier having high lipophilic and high proteinophilic affinity toensure uniform component suspension and prolonged antigenic release. 2.The vaccine of claim 1, said killed suspension having at least 2×10⁷bacteria.
 3. The vaccine of claim 1, said killed suspension having atleast 1×10¹⁰ bacteria.
 4. The vaccine of claim 1, said immune modulatorbeing present in an mount of at least 100 micrograms.
 5. The vaccine ofclaim 1, said protein and lipid binding carrier selected from the groupconsisting of fatty acids, oil based and alum based adjuvants.
 6. Thevaccine of claim 1, wherein said protein and lipid binding carrierfurther comprises a protein and lipid adjuvant of 1.5% by volumedialuminum trioxide.
 7. An immunization method for protecting an animalagainst endotoxin mediated, disseminated intravascular coagulation whichcomprises inoculating the animal with a vaccine which comprises a killedsuspension of a non-O-carbohydrate side chain bacterial mutant ATCC53000 devoid of sugars as a bacterin, a detoxified lipopolysaccharideimmune modulator having specific propensity for B-lymphocytes to causerapid proliferation of antibody progenitor cells and their earlieroccurrence, thus resulting in enhanced production of protective andneutralizing antibodies in a host's circulation against intact endotoxinand bacteria, and a protein and lipid binding carrier having highlipophilic and high proteinophilic affinity to ensure uniform componentsuspension and prolonged antigenic release.
 8. The immunization methodas in claim 7, wherein said bacterial mutant is produced by the processcomprising:inoculating a sterile, enriched broth with a bacterial mutantATCC 53000; inoculating said broth aerobically at approximately 37degrees C. for a maximal bacterial mass; killing said bacteria with abactericidal agent; washing said bacteria; and reconstituting saidbacteria for a preselected concentration.
 9. The process as in claim 8,said killing step comprising killing bacteria with the bactericidalagent Merthiolate.
 10. The process as in claim 8, said washing stepcomprising washing said bacteria with sterile, non-pyrogenic physiologicsaline.
 11. The immunization method as in claim 7, said immune modulatorproduced by the process comprising:admixing Gram negative bacterialendotoxin with pyridine-formic acid solution; methylating said admixtureby total reflux distillation; precipitating said methylatedlipopolysaccharide endotoxin admixture with alcohol; centrifuging thealcohol and methylated lipopolysaccharide endotoxin admixture; andmixing the precipitate with distilled water to a preselectedconcentration of lipopolysaccharide endotoxin.
 12. The process as inclaim 8, said admixing step utilizing a concentration of2:1pyridine-formic acid solution.
 13. The process as in claim 8, saidmethylating step including increasing the temperature of the admixtureto the boiling point and refluxing to obtain optimal methylation oflipopolysaccharide endotoxin.
 14. The process as in claim 8, additionalsteps of said process comprising:suspending in alcohol the precipitateof said centrifuging step; and recentrifuging said suspension prior tosaid mixing step.
 15. The immunization method as in claim 7, said killedsuspension having at least 1×10⁷ bacteria.
 16. The immunization methodas in claim 7, said killed suspension having at least 1×10¹⁰ bacteria.17. The immunization method as in claim 7, wherein said immune modulatoris present in an mount of at least 100 micrograms.
 18. The immunizationmethod as in claim 17, wherein said immune modulator is present in anamount of 100 to 4000 micrograms.
 19. The immunization method as inclaim 7, wherein said carrier is present in an mount of at least 1milligram of carrier per 1.5 milligrams of protein present in saidbacterial mutant.
 20. The immunization method as in claim 19, saidcarrier comprising 1.5% by volume Al₂ O₃.